ABSTRACT Digestive cysteine proteinases have been isolated from plant-parasitic nematodes as well as coleopteran and hemipteran insects. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pathogens and pests. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants were evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans). A PinII-beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter. Constitutive GUS expression was observed in leaf and root vascular tissue, and in some plants, expression was observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS expression approximately twofold over 24 h. Inoculation with root-lesion nematodes resulted in localized GUS expression. Populations of root-lesion nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and one line containing the PinII::OC-II transgene were reduced 29 and 32%, respectively, compared with a transgenic control line. These results suggest that oryzacystatins have the potential to confer increased resistance to the root-lesion nematode in alfalfa.